The control of gastrointestinal (GI) motility requires a complex interaction between several cell types such as smooth muscle and enteric nerves. In recent years we have added interstitial cells of Cajal (ICC) to this complex system. ICC regulate smooth muscle function and loss of ICC is associated with several motility disorders. ICC networks are constantly turning over and maintenance of the networks requires tight regulation of ICC proliferation. Ano-1, a Ca2+ activated Cl- channel has recently been found to be selectively expressed on ICC in the muscle layers of the gut as well as in several tumors, including gastrointestinal stromal tumors. The overall objective of this proposal is to determine the role of Ano1 in ICC function. The central hypothesis of this proposal is that Ano-1 has dual, novel roles in ICC physiology. It acts as a key molecule in the regulation of ICC proliferation and as an ion channel required for the repolarization of the slow wave. The central hypothesis will be tested in two specific aims. Specific Aim 1 will test the hypothesis that Ano1 regulates proliferation of ICC and Specific aim 2 will test the hypothesis that Ano1 is required for repolarization of the slow wave. The first specific aim is supported by preliminary data that show that Ano1 in the tunica muscularis is expressed only on ICC, that Ano1 may be used to detect loss of ICC in motility disorders, that a lack of Ano1 is associated with a decrease in proliferation of ICC and that an increase in expression of Ano-1 is associated with an increase in ICC proliferation. The preliminary data also provide evidence, from bioinformatics analysis of microarray data comparing knockout to wild type mice, that key molecules associated with the control of proliferation, including CDK1/cdc2, are down-regulated when Ano1 is absent. The second specific aim is supported by preliminary data that show that Cl- channel blockers alter the duration of the plateau of the slow wave, that the slow wave is prolonged when Ano-1 expression is decreased and that it normalizes after Ano-1 is expressed. The PI will test the central hypothesis by a combination of innovative techniques including 3D reconstructions of immunohistochemical data, Western blots, RT-PCR, single cell PCR, quantitative PCR, lentivirus and si/shRNA knock down techniques, electroporation, knockout mice, organotypic and single cell cultures, bioinformatic analysis of microarray data, proteomics, Ca2+ and Cl- imaging, microelectrode recordings and patch clamp techniques. Successful completion of the proposed studies has both basic significance and clinical impact. Our work on the newly discovered protein Ano1 will provide mechanistic information on both how ICC networks are maintained and on the regulation of the slow wave. Of immediate clinical significance our work will inform clinicians and pathologists on use of antibodies to Ano1 to assess ICC in motility disorders and provide targets for future therapeutic interventions. Our work also has broad implications beyond the GI tract. As Ano-1 is expressed in many organs and tumors including gastrointestinal stromal tumors, our findings will likely apply to several other organs outside of the GI tract.